Therapeutic Potential of Phytocannabinoid Cannabigerol for Multiple Sclerosis: Modulation of Microglial Activation In Vitro and In Vivo

Multiple sclerosis (MS) is a widespread chronic neuroinflammatory and neurodegenerative disease. Microglia play a crucial role in the pathogenesis of MS via the release of cytokines and reactive oxygen species, e.g., nitric oxide. Research involving the role of phytocannabinoids in neuroinflammation is currently receiving much attention. Cannabigerol is a main phytocannabinoid, which has attracted significant pharmacological interest due to its non-psychotropic nature. In this research, we studied the effects of cannabigerol on microglial inflammation in vitro, followed by an in vivo study. Cannabigerol attenuated the microglial production of nitric oxide in BV2 microglia and primary glial cells; concomitant treatment of the cells with cannabigerol and telmisartan (a neuroprotective angiotensin receptor blocker) decreased nitric oxide production additively. Inducible nitric oxide synthase (iNOS) expression was also reduced by cannabigerol. Moreover, tumor necrosis factor-α (TNF-α), a major cytokine involved in MS, was significantly reduced by cannabigerol in both cell cultures. Next, we studied the effects of cannabigerol in vivo using a mice model of MS, experimental autoimmune encephalomyelitis (EAE). The clinical scores of EAE mice were attenuated upon cannabigerol treatment; additionally, lumbar sections of EAE mice showed enhanced neuronal loss (relative to control mice), which was restored by cannabigerol treatment. Altogether, the set of experiments presented in this work indicates that cannabigerol possesses an appealing therapeutic potential for the treatment of MS.


Introduction
Multiple sclerosis) MS) is the most prevalent chronic neuroinflammatory disease. It is an autoimmune and neurodegenerative disease defined by neuroinflammation, demyelination, axonal loss and neurodegeneration [1][2][3]. Microglia play a role in inflammatory responses, homeostasis, and tissue regeneration. Microglia have contributed to the development of autoimmune encephalomyelitis (EAE), in animal models of MS already in the initial stages [4]. Activated microglia present antigens and secrete major cytokines such as tumor necrosis factor-α (TNF-α). Additionally, they are involved in the demyelination and phagocytosis of the degraded myelin [5].
Studies showed that detected neurodegeneration is linked with acute demyelinating lesions [6][7][8], suggesting that inflammatory-related neuronal injury occurs early during the course of the disease. It has been recently shown that during experimental MS, a reversible form of focal axonal swelling, potentially evolving into axonal disruption, can be detected during the earlier phase of neuroinflammation, when the myelin sheet has not yet been
For each in-vitro experiment, serum-free medium (SFM) was added to the cells 4 h before the experiment initiation. Thereafter, microglia were treated with SFM containing Biomolecules 2023, 13, 376 3 of 12 0.1% bovine serum albumin (BSA) and HEPES buffer (10 mM at pH 7.4) in the absence or presence of test agents for 22 h. All culture media were purchased from Biological Industries (Kibbutz Beit-Haemek, Israel). CBG, LPS from Escherichia coli O55:B5 and poly-L-lysine were purchased from Sigma Aldrich (Rehovot, Israel).
Primary neonatal rat glial cell cultures were prepared from whole brains of 1-day-old Wistar rats, and grown according to accepted protocols [23,24]. Immunocytochemistry studies as previously described [24,25] revealed that these cultures contain about 80% astrocytes and about 20% microglia.

Cell Viability
Cells were seeded at a density of 1 × 10 4 cells per well in 96-well plates and cultivated overnight in complete RPMI-1640 medium. Thereafter, cells were treated with the respective test agents, as described above. Subsequently, XTT reagent was mixed with the activation reagent, at a ratio of 50:1 according the manufacture's protocol (Biological Industries, Kibbutz Beit-Haemek, Israel) and was added to each well in a 1:2 ratio. Absorbance was measured at 450 nm against a reference wavelength at 650 nm after 1 h incubation at 37 • C using a microplate reader (Model 680, Bio-Rad, Hercules, CA, USA).

Determination of NO Levels (Griess Reaction)
Nitrite levels in culture media, as an indicator for NO release, were determined by an established assay using Griess reagent (Sigma Aldrich, Rehovot, Israel).

Determination of TNF-α Levels (ELISA)
TNF-α levels in the culture media were determined using enzyme-linked immunosorbent assay (ELISA) kit (BD Biosciences, San Diego, CA, USA) according to the manufacturer's protocol.

Western Blot Analysis
Forty (40) µg of protein from whole cell lysate were loaded on 7.5% polyacrylamide-SDS gels and blotted on a nitrocellulose membrane. After blocking with 4% BSA for 90 min, membranes were incubated overnight at 4 • C with rabbit anti-iNOS antibody (130 kDa) (1:500, Cayman Chemicals, Ann Arbor, MI, USA). Upon washing, the blots were incubated for 90 min in the corresponding conjugated donkey anti-rabbit antibody (1:10,000, GE Healthcare, Buckinghamshire, UK). The position of the individual protein was detected after exposure to the ChemiDocTM XRS+ (Bio-Rad Laboratories, Hercules, CA, USA) image system. Band-intensity analysis was performed using a computerized image analysis system (ImageJ software, version 1.40C, NIH). Protein quantity was normalized to β-actin protein (40 kDa) level measurements using mouse monoclonal anti-β-Actin−Peroxidase antibody (1:20,000, Sigma Aldrich, Israel).

Active MOG-Induced EAE Model
Female eight-week-old C57BL/6 mice (Envigo, Jerusalem, Israel) were immunized with myelin oligodendrocyte glycoprotein (MOG) [peptide  (AnaSpec, Fremont, CA, USA). Each mouse was injected subcutaneously (s.c.) into 2 sites on the back, adjacent to each of the hind limbs (total volume 200 µL), with 200 µg MOG emulsified with a mixture of 200 µg/mL killed Mycobacterium tuberculosis H37RA (Difco, Detroit, MI, USA) in complete Freund's adjuvant (BD Biosciences, San Jose, CA, USA). Thereafter, each animal was injected intraperitoneally (i.p.) with 400 ng/mL reconstituted pertussis toxin (ENCO, Petah Tikva, Israel), which was repeated two days after the initial immunization. National and institutional guidelines for the care and use of laboratory animals were followed.
After immunization, the mice were evaluated for neurological scores as follows: 0 normal; 0.5 mild ataxia of the hind limb; 1 decreased tail tone; 1.5 righting reflex within 3 s; 2 righting reflex between 4 and 7 s; 2.5 righting reflex between 7 and 10 s; 3 hind limbs paralysis or absolute loss of righting reflex; 4 front and hind limbs paralysis (n = 12, Biomolecules 2023, 13, 376 4 of 12 6 of them obtained only MOG and 6 of them obtained MOG+CBG). From day 12 postimmunization treatment, administrations were performed i.p. for four consecutive days. Control EAE mice received only the vehicle solution composed of Tween-20:ethanol:saline at a ratio of 1:1:8 (n = 4). CBG EAE mice received CBG (10 mg/kg) dissolved in the vehicle solution (n = 6). The experiment was terminated by euthanizing the mice, followed by cardiac perfusion. Thereafter, spinal columns were fixed in 4% formaldehyde at 4 • C overnight and cryoprotected in 20% sucrose for 48 h at 4 • C. Then, spinal cords were dissected and mounted in OCT (Scigen Scientific, Gardena, CA, USA), snap frozen at −40 • C and, finally, stored at −80 • C. Sections were mounted on slides with Immu-Mount (Thermo Scientific, Ann Arbor, MI, USA). Images were obtained using the Olympus FluoView FV1000 confocal microscope (Olympus, Hamburg, Germany).

Statistical Analysis
Experimental data are presented as the mean ± standard error of mean (SEM). For significance assessment between groups, one-way analysis of variance (ANOVA) and posthoc multiple comparison test (Tukey-Kramer multiple comparison test) were performed. Statistical significance was considered at p < 0.05.

Cell Viability
We examined the viability of BV2 microglial cells treated with concentrations of 1, 5, 10 and 25 µM CBG. While 1 µM CBG significantly increased cell viability as compared to control or DMSO-treated cells, CBG at 5, 10 µM did not change the viability of BV2 cells. The highest concentration of CBG used, namely, 25 µM, attenuated cell viability by 70%. Almost a complete reduction in cell viability was observed using the transcription inhibitor actinomycin D ( Figure 1A). No significant differences in cell viability were found between control and LPS-treated (0.5 µg/mL) with or without CBG treatment in primary rat glial cells ( Figure 1B).

NO Release and TNF-α Production
Treatment with LPS (7 ng/mL) stimulated NO release from BV2 cells into the culture media, while CBG dose dependently (5, 10 µM) reduced LPS-induced NO production ( Figure 2A). Without LPS induction, the basal NO level was significantly induced by 5 µM CBG. NO levels were significantly reduced when CBG concentration was increased (10 µM) (Figure 2A). LPS-induced TNF-α synthesis was significantly decreased by 5 µM CBG ( Figure 2B). In addition to BV2 microglia, also in rat primary mixed glial cells, LPS (0.5 µg/mL) significantly induced NO and TNF-α production. Treatment with 5 µM CBG reduced significantly the NO release ( Figure 2C), while TNF-α production was attenuated by 13% after the application of 5 µM CBG ( Figure 2D). Moreover, we examined the synthesis of NO in BV2 cells stimulated by LPS (7 ng/mL) and treated with the neuroprotective agents telmisartan (5 µM) ( Figure 3A) or captopril (1 mM) ( Figure 3B) with or without CBG (5 µM) (A). While LPS significantly enhanced NO synthesis as compared to non-stimulated cells (control), telmisartan (5 µM) and CBG (5 µM) attenuated this effect by 17% and 29%, respectively. Concomitant CBG and telmisartan treatment ( Figure 3A) additively decreased NO production in LPS-induced BV2 microglia. Concomitant CBG and captopril treatment decreased NO production as well; this effect was higher than the effect of each agent separately, but was lower than the additive degree ( Figure 3B). and rat primary mixed glial cells (B) were pre-incubated with SFM for 4 h. Then, CBG was added for 22 h. Cell viability was determined by XTT proliferation assay. Data are presented as means ± SEM and are representatives of two independent experiments (n = 6). Statistical significance was determined using one-way ANOVA, followed by a Tukey-Kramer multiple comparison test. ** p < 0.01 vs. control; *** p < 0.001 vs. control; ^ p < 0.05 vs. DMSO; ^^^ p < 0.001 vs. DMSO.

NO Release and TNF-α Production
Treatment with LPS (7 ng/mL) stimulated NO release from BV2 cells into the culture media, while CBG dose dependently (5, 10 μM) reduced LPS-induced NO production ( Figure 2A). Without LPS induction, the basal NO level was significantly induced by 5 µM CBG. NO levels were significantly reduced when CBG concentration was increased (10 μM) (Figure 2A). LPS-induced TNF-α synthesis was significantly decreased by 5 µM CBG ( Figure 2B). In addition to BV2 microglia, also in rat primary mixed glial cells, LPS (0.5 µg/mL) significantly induced NO and TNF-α production. Treatment with 5 µM CBG reduced significantly the NO release ( Figure 2C), while TNF-α production was attenuated by 13% after the application of 5 µM CBG ( Figure 2D). Moreover, we examined the synthesis of NO in BV2 cells stimulated by LPS (7 ng/mL) and treated with the neuroprotective agents telmisartan (5 μM) ( Figure 3A) or captopril (1 mM) ( Figure 3B) with or without CBG (5μM) (A). While LPS significantly enhanced NO synthesis as compared to non-stimulated cells (control), telmisartan (5 μM) and CBG (5 μM) attenuated this effect by 17% and 29%, respectively. Concomitant CBG and telmisartan treatment ( Figure 3A) additively decreased NO production in LPS-induced BV2 microglia. Concomitant CBG and captopril treatment decreased NO production as well; this effect was higher than the effect of each agent separately, but was lower than the additive degree ( Figure 3B).

iNOS Protein Levels
The 22 h exposure to LPS in BV2 cells (7 ng/mL) or primary glial cells (0.5 µg/mL) resulted in a robust increase in iNOS protein levels (Figure 4), by more than 90% as compared with the control. Yet, 22 h incubation with LPS together with 5 µM CBG significantly reduced iNOS expression levels by 65% in BV2 cells ( Figure 4A,B) and by 45% in primary rat glial cells ( Figure 4C,D), respectively. Higher application of 10 µM CBG in the primary rat glial culture reduced iNOS level by 50%. CBG alone did not alter iNOS protein expression level.

iNOS Protein Levels
The 22 h exposure to LPS in BV2 cells (7 ng/mL) or primary glial cells (0.5 µg/mL) resulted in a robust increase in iNOS protein levels (Figure 4), by more than 90% as compared with the control. Yet, 22 h incubation with LPS together with 5 µM CBG significantly reduced iNOS expression levels by 65% in BV2 cells ( Figure 4A,B) and by 45% in primary rat glial cells ( Figure 4C,D), respectively. Higher application of 10 µM CBG in the primary rat glial culture reduced iNOS level by 50%. CBG alone did not alter iNOS protein expression level.

EAE Studies
In vivo, we investigated the potential of i.p. CBG at a clinically relevant dose on neurological scores of EAE mice ( Figure 5). Immunization with MOG induced EAE in mice, giving a mean onset on day 9 post-immunization (p.i.). All MOG-treated mice developed a disease with a mean score of 1.75. By contrast, the clinical manifestations of EAE were attenuated in mice receiving four injections of CBG (10 mg/kg) at days 12 to 15 upon immunization) Figure 5). In the mice that received CBG, a first peak in disease severity appeared between days 12 and 13 with a subsequent decline until day 17 postimmunization. Thereafter, a second peak followed at day 18 which slowly declined until the end of the experiment. The mean severity score in the group receiving CBG was always less than 1 throughout the course of the experiment from day 9 to day 26.
The 22 h exposure to LPS in BV2 cells (7 ng/mL) or primary glial cells (0.5 µg/mL) resulted in a robust increase in iNOS protein levels (Figure 4), by more than 90% as compared with the control. Yet, 22 h incubation with LPS together with 5 µM CBG significantly reduced iNOS expression levels by 65% in BV2 cells ( Figure 4A,B) and by 45% in primary rat glial cells ( Figure 4C,D), respectively. Higher application of 10 µM CBG in the primary rat glial culture reduced iNOS level by 50%. CBG alone did not alter iNOS protein expression level.

EAE Studies
In vivo, we investigated the potential of i.p. CBG at a clinically relevant dose on neurological scores of EAE mice ( Figure 5). Immunization with MOG induced EAE in mice, giving a mean onset on day 9 post-immunization (p.i.). All MOG-treated mice developed a disease with a mean score of 1.75. By contrast, the clinical manifestations of EAE were attenuated in mice receiving four injections of CBG (10 mg/kg) at days 12 to 15 upon immunization ( Figure 5). In the mice that received CBG, a first peak in disease severity appeared between days 12 and 13 with a subsequent decline until day 17 post-immunization. Thereafter, a second peak followed at day 18 which slowly declined until the end of the experiment. The mean severity score in the group receiving CBG was always less than 1 throughout the course of the experiment from day 9 to day 26.

Immunohistochemistry Evaluations
The effects of i.p. administration of CBG on astrocytosis, demonstrated by GFAP staining (Figure 6a-c,g); microgliosis, demonstrated by Iba1 staining (Figure 6d-f,h,i); and neuronal loss, demonstrated by NeuN staining (Figure 7a-c,g) and CD4 staining ( Figure  7d-f,h) were investigated in non-treated and MOG-treated (EAE) mice by immunohistochemistry in lumbar sections of spinal cords. Lumbar sections of control mice showed low GFAP, Iba1 and CD4 staining. By contrast, lumbar sections of EAE mice exhibited induced levels of astrogliosis, microglial activation and CD4 expression when compared to control mice. As expected, neuronal loss was enhanced in EAE mice as compared with control mice, but was restored by 10 mg/kg CBG when administrated for four consecutive days. The same CBG treatment significantly reduced the areas stained for GFAP in EAE mice, while CD4 and Iba1 expression was not changed upon CBG treatment.

Immunohistochemistry Evaluations
The effects of i.p. administration of CBG on astrocytosis, demonstrated by GFAP staining (Figure 6a-c,g); microgliosis, demonstrated by Iba1 staining (Figure 6d-f,h,i); and neuronal loss, demonstrated by NeuN staining (Figure 7a-c,g) and CD4 staining (Figure 7d-f,h) were investigated in non-treated and MOG-treated (EAE) mice by immunohistochemistry in lumbar sections of spinal cords. Lumbar sections of control mice showed low GFAP, Iba1 and CD4 staining. By contrast, lumbar sections of EAE mice exhibited induced levels of astrogliosis, microglial activation and CD4 expression when compared to control mice. As expected, neuronal loss was enhanced in EAE mice as compared with control mice, but was restored by 10 mg/kg CBG when administrated for four consecutive  Immunodetections were quantified and plotted as integrated density for each antibody (g,h). One way ANOVA followed by a Tukey-Kramer multiple comparison test were performed to determine statistical significance. *** p < 0.001 vs. control; ^p < 0.05 vs. MOG. The scale bar is 50 µm. Representative lumbar layers from the mice groups are presented (n = 4-6 for each group). Immunodetections were quantified and plotted as integrated density for each antibody (g,h). One way ANOVA followed by a Tukey-Kramer multiple comparison test were performed to determine statistical significance. *** p < 0.001 vs. control;ˆp < 0.05 vs. MOG. The scale bar is 50 µm.

Discussion
MS is accompanied by activation of glia [26]. In fact, microglia play a dual role, sometimes inducing inflammation, but in other cases inducing repair by clearing myelin and cell debris [27,28]. Astrocytes are also a major component of MS plaques [29] well positioned to enhance inflammation by cytokines such as TNF-α and free radicals such as NO, but they may also limit damage by providing metabolic support to axons [30].
Glial NO can rapidly react with a superoxide anion to form peroxynitrite (ONOO -), one of the most deleterious reactive oxygen species [31]. Peroxynitrite plays an important role in the pathology of demyelinating diseases, such as MS [32][33][34].
In the present study, we demonstrate the anti-inflammatory and anti-oxidative effects of CBG, by itself, as revealed by the attenuation of BV2 microglial production of NO, iNOS and TNF-α stimulated by LPS (7 ng/mL) in BV2 cells and in primary glial cultures induced by LPS (0.5 µg/mL). CBG in both models of inflammation demonstrated higher (two-five-fold) potency in attenuating microglial inflammatory response as compared to data published previously [21]. In recent years, a series of CBG quinone derivatives such as VCE-003, which act as PPAR-gamma activators, showing low affinity for cannabinoid receptors, have been characterized [21,35]. As shown by Gugliandolo et al., VCE-003 reduced the expression of the iNOS protein in LPS and IFN-γ-treated BV2 microglia [21]. In addition, VCE-003.2 reduced iNOS mRNA in BV2 microglia exposed to high levels of LPS [22]. Interestingly, CBG such as CBD may also be converted to CBG-hydroxyquinone (a precursor of VCE-003.2) during liver metabolism, explaining at least part of the neuroprotective effect of CBG in vivo.
Female mice were chosen to be studied in this EAE model, since autoimmune diseases are more prevalent in females than males. This discrepancy was also found in animal models. Increased spinal-cord lesions and demyelination are shown in female EAE mice vs. males.
This study provides evidence that CBG by itself attenuated the neurological deficit score in EAE mice. It also reduced astrogliosis and decreased neuronal loss in EAE mice. An increase in CD4 T-cell populations was also observed upon the CBG treatment of EAE mice. The major pro-inflammatory CD4 T-cells associated with autoimmune diseases, including MS, are the Th1 CD4 T-cells. These cells secrete IFN-γ and TNF-α [36,37]. Autoimmune diseases are also associated with Th2 CD4 T-cells, which are induced by IL-4 [38]. Th2 CD4-related cytokines, such as IL-4 and IL-10, are anti-inflammatory and improve symptoms in MS patients. Th1 CD4 cytokines have been shown to increase inflammation, and lead to disease progression and the worsening of symptoms [39,40]. Th2 and Th1 cytokines can cross-inhibit each other and the progression of disease may depend on the balance between both types of cytokines [39,40]. In this study, we saw an improvement in EAE symptoms, suggesting the occurrence of a shift toward a Th2 cytokine anti-inflammatory response. The protective effects observed here may also be due to changes in microglial inflammatory functions. An analysis of the CD4+ T cell phenotypes in EAE spinal cords would help determine the anti-inflammatory effects of CBG. Our data correlates with other studies using different in-vivo models. Valdeolivas has shown the anti-inflammatory role of CBG in two experimental models of Huntington's disease [41]. Beneficial effects of CBG were also shown in experimental inflammatory bowel disease [42]. VCE-003 alleviated neuroinflammation and motor deficits in the viral TMEV model of MS. VCE-003 also suppressed immune responses and neuroinflammation in EAE mice [35]. In summary, based on its antioxidant and anti-inflammatory activities, CBG may hold great promise as an antioxidant agent and, therefore, may be used in clinical practice as a new approach in oxidative-stress-related disorders. An additional intriguing point was the potential therapeutic interest in the combined administration of telmisartan/captopril and CBG. ARBs such as telmisartan and ACEIs such as captopril were shown to be neuroprotective by us and others. Both telmisartan and captopril in combination with CBG significantly reduced NO synthesis compared to each compound by itself. Telmisartan, but not captopril, acted additively with CBG to inhibit NO production in LPS-stimulated microglia. This suggests that distinct cellular signaling pathways in microglia may be activated by telmisartan and captopril. Further work is warranted to determine the mechanism of action of CBG and the compounds tested here, at both receptor and intracellular signaling levels, to enhance our ability to develop novel safe and effective treatment strategies for microglial inflammation and neurodegeneration.  Institutional Review Board Statement: The animal study protocol was approved by the Ben-Gurion University of the Negev Animal Use and Care Committee (Protocol IL-64-08-2020).

Data Availability Statement:
The data presented in this study are available on request from the corresponding author. The data are not publicly available due to privacy issues.